Ann Clin Biochem OnlineFirst, published on July 13, 2015 as doi:10.1177/0004563215590451

Review Article Annals of Clinical Biochemistry 0(0) 1–6 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0004563215590451 acb.sagepub.com

Lipoprotein lipase and atherosclerosis Junji Kobayashi1 and Hiroshi Mabuchi2

Abstract Lipoprotein lipase has long been known to hydrolyse triglycerides from triglycerides-rich lipoproteins. More recently, it has been shown to promote the binding of lipoproteins to various lipoprotein receptors. Evidence is also presented regarding the possible atherogenic role of lipoprotein lipase. In theory, lipoprotein lipase deficiency should help to clarify this question. However, the rarity of this condition means that it has not been possible to conduct epidemiological studies. An alternative approach is to investigate the correlation of lipoprotein lipase with onset of cardiovascular disease in prospective studies in large population-based cohorts. Complementary with this approach, animal models have been used to explore the atherogenicity of lipoprotein lipase expressed by macrophages.

Keywords Atherosclerosis, lipoprotein lipase Accepted: 15th May 2015

Introduction Lipoprotein lipase (LPL) (EC 3.1.1.34) is synthesized and secreted in several tissues, such as adipose tissue, skeletal muscle, cardiac muscle and macrophages (M), binding to the vascular endothelial cell surface of the capillary through heparan sulphate. LPL is a member of the lipase gene family, which includes pancreatic lipase, hepatic lipase and endothelial lipase.1–3 The human LPL gene is located on the short arm of chromosome 8 (8p22), is about 35 kb in length, contains 10 exons and encodes an enzyme protein consisting of 448 amino acids.2,4,5 LPL catalyses hydrolysis of triglycerides (TG) from chylomicrons and very low density lipoproteins (VLDL), facilitating the incorporation of free fatty acids into adipocytes,6,7 where they are resynthesised into TG and stored. They are utilized as an energy source in cardiac and skeletal muscle. The NH2-terminal domain of LPL controls its catalytic properties, whereas the COOH-terminal domain modulates substrate specificity.8,9 In the 1990s, a number of researchers established that LPL has an additional

bridging function, promoting the binding of different lipoproteins to their receptors.10–14 The dual roles of LPL – promoting lipoprotein metabolism and acting as a ligand for binding of lipoproteins to their receptors – have led to speculation about its possible role in the onset of atherosclerosis. Various approaches have been used to study this role. For example, LPL deficiency provides an obvious means of examining this question. However, this condition is rare (approximately one per million in most populations) and it is not possible to conduct 1 General Internal Medicine, Kanazawa Medical University, Uchinada, Daigaku, Ishikawa, Japan 2 Lipid Research Course, Kanazawa University Graduate School of Pharmaceutical, Health Sciences, Kakuma-machi, Kanazawa, Ishikawa, Japan

Corresponding author: Junji Kobayashi, General Internal Medicine, Kanazawa Medical University, Uchinada Daigaku 1-1, Ishikawa Prefecture, Japan. Email: [email protected]

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epidemiological studies. Experimental studies have been performed using animal models to explore the atherogenicity of LPL expressed by M. We review here the evidence that LPL plays a role in the onset of atherosclerosis.

Studies which indicate that LPL is proatherogenic LPL is expressed in atherosclerotic lesions It was first reported in the 1990s that LPL in atherosclerotic lesions was derived from M;15,16 moreover, differences in M expression of LPL contributed to differences in the development of atherosclerotic plaque formation.17 Concentrations of LPL protein, activity and mRNA in atherosclerosis-prone mice were found to be much higher (several-fold) than in atherosclerosis-resistant counterparts. Babaev et al.18 transplanted fetal liver cells (from which M cells are derived) from LPL þ/þ mice, LPL /þ mice and LPL / mice into irradiated female mice and examined atherosclerotic lesions of the aorta. Mice transplanted with fetal liver cells from LPL þ/þ mice had the most severe atherosclerotic lesions in the aorta, suggesting that M LPL may contribute to the severity of atherosclerotic lesions. Ichikawa et al.19 compared atherosclerotic lesions in rabbits with over-expressed Mspecific human LPL, with lesions in wild-type strains, after giving both groups food containing 0.3% cholesterol. Serum lipids were comparable but atherosclerotic lesions were more prominent in the former than in the latter. In a similar vein, peritoneal M from Mspecific LPL / mice crossed with apo E / mice showed less susceptibility to foam cell formation compared with those from apo E / mice,20 suggesting that M LPL may promote atherosclerosis formation.

Elevated LPL activity and mass in post-heparin plasma in subjects with advanced atherosclerosis Investigators at the National Institutes of Health examined the volume of total calcific atherosclerosis in the heart and thoracic aorta and coronary artery calcific atherosclerosis in 15 patients with homozygous familial hypercholesterolemia.21 Both LPL activity and mass correlated with these parameters.

LPL is involved in the formation of atherogenic lipoproteins as an enzyme LPL hydrolyses TG from chylomicrons and VLDL forming chylomicron remnants and VLDL remnants. These are rich in cholesterol esters22 and become incorporated into M in vitro.23 Furthermore, the free fatty

acids formed by this LPL action are re-esterified by M.24 This process promotes cholesterol ester accumulation in M, leading to the transformation of M into foam cells. LPL also converts VLDL into intermediatedensity lipoproteins (IDL), converted in turn into low density lipoproteins (LDL) by the action of hepatic lipase. LDL is oxidized in vascular subendothelium, incorporated into M via the scavenger receptor, again contributing to formation of foam cells.25

LPL function as a ligand, independent of its enzyme activity In 1991, Beisiegel et al.10 reported that human and bovine LPL promote binding of chylomicrons to a lipoprotein receptor-related protein (LRP) on the surface of HepG2 cells, independently of its enzyme activity. LPL was subsequently shown to be involved in binding a variety of lipoproteins to individual receptors.11,12 The interaction of LDL with LPL is found to be enhanced by its oxidization.13,14 By these mechanisms LPL promotes lipoprotein accumulation in the arterial subendothelial matrix leading to atherosclerosis. LPL not only promotes binding of lipoproteins to receptors but also promotes adhesion of monocytes to the vascular endothelium.26,27

Studies which indicate that LPL is antiatherogenic LPL deficiency and atherosclerosis In LPL deficiency, the elevated lipoproteins (chylomicrons) are too large to penetrate into the vascular endothelium, and plasma LDL cholesterol concentrations are reduced; as a result, development of atherosclerosis is attenuated.7 Consistent with this, Ebara et al.28 reported a 66-year-old female with LPL deficiency without any obvious atherosclerotic disease in carotid, femoral and coronary arteries as assessed by ultrasonography and electrocardiography after exercise-tolerance testing. They also reported a 60-yearold female with LPL deficiency (caused by a different mutation), who had no atherosclerotic lesions in her coronary arteries as examined by multidetector computer tomography (MDCT).29 We have similarly reported a 53-year-old man with LPL deficiency without atherosclerosis.30 On the other hand, Benlian et al.31 investigated four patients with profound functional deficiency of LPL associated with reduced enzymatic mass due to missense mutations on both alleles of the LPL gene; in all four, premature atherosclerosis (before age 55) was observed. Similarly, Saiki et al.32 reported a 55-year-old man with LPL deficiency associated with coronary

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Kobayashi and Mabuchi

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Table 1. Literature referring to lipoprotein lipase deficiency in atherosclerosis. Date

Reference 31

1996 1996 1996 1996 2001 2003 2005 2007

Benlian et al. Benlian et al.31 Benlian et al.31 Benlian et al.31 Ebara et al.28 Saika et al.32 Kawashiri et al.30 Ebara et al.29

Age and sex

LPL mutation

Atherogenic

52 y 54 y 67 y 62 y 66 y 55 y 53 y 60 y

G118R/G118R G118E/R243C Frameshift/L286P T101A/D250N Y61X/Yg1/X L303F/L303F delG916/delG916 G118E/G118E

Yes Yes Yes Yes No Yes No No

male male male female female male male female

LPL: lipoprotein lipase.

A

B

o-Phenylenediamine

Tetramethylbenzidine

HRP

HRP G Goat anti-chicken IgG cconjugated with HRP Chicken anti-bovine milk LPL PoAb

Mouse anti-human LPL MoAb 88B8

LPL

LPL

Mouse anti-bovine milk LPL MoAb

Mouse anti-human LPL MoAb 57A5

plate

plate

Figure 1. (a) ELISA for quantifying human LPL protein concentration in either serum or PHP using antibovine milk LPL monoclonal antibody and antibovine milk LPL polyclonal antibody.44 (b) ELISA for quantifying human LPL protein concentration using two different monoclonal antibodies against human LPL (57A5 and 88B8) for the sandwich ELISA.45 MoAb: monoclonal antibody. ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; LPL: lipoprotein lipase; HRP: horseradish peroxidase; o: ortho; PoAb: polyclonal antibody; MoAb: monoclonal antibody.

artery disease and severe systemic atherosclerosis. Furthermore, in heterozygous LPL deficiency, reduced LPL activity is associated with premature atherosclerosis33,34 or the onset of familial combined hyperlipidemia.35 Finally, Hu et al.36 reported in a meta-analysis that LPL Asn291Ser mutations were associated with high TG, low HDL and high rates of coronary artery disease. Conversely, LPL mutations leading to increased LPL activity were protective against the development of coronary artery disease in another meta-analysis.37 Table 1 summarizes the reports concerning LPL deficiency and atherosclerosis. The nature of the association between LPL deficiency and atherosclerosis is

unclear. Some LPL-deficient individuals have mutations in the gene of apolipoprotein A–V,38 another molecule involved in TG metabolism, further complicating the question of whether or not LPL deficiency is proatherogenic or anti-atherogenic. Animal models of LPL deficiency have been used to examine this issue. Zhang et al.39 studied LPL-deficient mice, where human LPL gene was introduced at birth with adenoviral vectors. The mice exhibited low HDL-C and marked hypertriglyceridaemia on a normal chow diet. Although at four months of age there were no atherosclerotic lesions of the aorta in the LPL-deficient mice but at 15 months of age more advanced atherosclerotic lesions were observed compared with those in wild-type

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or with heterozygous LPL deficiency. These findings suggest that as individuals with LPL deficiency age, atherosclerotic lesions may progress. The authors of this study proposed that chylomicron derived from LPL-deficient subjects may contribute to increasing the expression of VCAM-1 and MCP-1 from endothelial cells.

Atherosclerosis in animal models over-expressing LPL In animal models, over-expressing human LPL is associated with improved serum lipid profile.40–42 Shimada et al.41 found that over-expression of human LPL in LDL receptor / mice was associated with a significant reduction of aortic atherosclerotic lesions, as well as decreases in TG and remnants in plasma, compared with LDL receptor/ mice without human LPL over-expression. Tsutsumi et al.43 found that NO-1886, a compound that increases LPL activity, was associated with elevated HDL-C and reduced TG, and that its long-term (90-day) administration inhibits atherogenesis in the coronary arteries of rats Table 2. A multiple regression analysis on the relationship of intima media thickness of common carotid artery assessed by ultrasonography to LPL, age, gender, body mass index, LDLC, HDLC and TG.

Ln LPL concentration Age Gender Body mass index LDL-cholesterol HDL-cholesterol Triglycerides

SE



t

P

0.142 0.004 0.092 0.020 0.001 0.003 0.0004

0.461 0.409 0.093 0.091 0.296 0.046 0.215

2.21 2.47 0.519 0.497 1.608 0.224 0.896

0.0352 0.0198 0.608 0.622 0.119 0.825 0.378

SE: standard error; : beta; t: t-statistic; P: P value; LPL: lipoprotein lipase; HDLC: high-density lipoprotein cholesterol; TG: triglycerides. Data from Kobayashi et al.45

with experimental atherosclerosis. Fan et al.42 found that when transgenic rabbits expressing human LPL were fed a cholesterol-rich diet, the development of hypercholesterolaemia and aortic atherosclerosis was dramatically suppressed. Thus, systemically increased LPL activity affects the metabolism of all classes of lipoproteins, and plays a crucial role in plasma TG hydrolysis and lipoprotein conversion. Over-expression of LPL appears to protect against diet-induced hypercholesterolaemia and atherosclerosis.

Studies suggesting serum LPL protein concentration is a useful biomarker predicting cardiovascular disease In clinical practice, LPL used to be quantified by measuring its activity in post-heparin plasma (PHP) using isotope-labelled substrate. In 1993, we established a sandwich enzyme-linked immunoassay (EIA) system for quantifying LPL protein concentration in PHP, using antibovine milk LPL monoclonal antibody and antibovine milk LPL polyclonal antibody (Figure 1(a)).44 Subsequently, the clinical significance of measuring LPL concentrations in serum rather than PHP was clarified.45,46 Hanyu et al.47 found that serum LPL mass correlated significantly with insulin sensitivity analysed by minimal modelling, regardless of whether the subjects had normal glucose tolerance, impaired glucose tolerance or diabetes. Hitsumoto et al.48,49 found that men with coronary atherosclerosis had significantly lower pre-heparin LPL mass than did men without atherosclerosis or healthy men. Thus, LPL mass appears to be an independent determinant of coronary artery disease48,49 even after adjusting for metabolic parameters, including serum TG and HDL-C. We examined the correlation of intimamedia thickness (IMT) of the carotid artery by ultrasonography and serum LPL concentration in dyslipidemic patients. There was an inverse correlation between serum LPL concentration and IMT,

Table 3. Odds ratios for future CAD according to serum LPL concentration quartile. LPL quartile

1

2

3

4

Range (mg/L) n Model 1 Model 2 Model 3 Model 4

91 217/496 0.06 (0.53 0.77 (0.60 0.88 (0.67 0.87 (0.67

to to to to

1.0) 1.17) 1.23) 1.21)

to to to to

0.83) 0.94) 1.03) 1.02)

P

to to to to

0.83) 0.99) 1.14) 1.13